Metal ions such as calcium are involved in many cellular processes including signal transduction. Small variances in intracellular ion levels can have a major impact on cellular processes. Measurement of ion levels provides a very sensitive method for identifying various cellular activities.
Several fluorescent calcium indicators known in art are employed in biological research and high throughput screening. Generally, long wavelength indicators, such as rhodamine-based compounds bear a positive charge. Positively charged molecules compartmentalize in cell mitochondria. Because calcium ion release in activated cells happens in the cytosol, positively charged indicators show a weak response to calcium ion influx. Alternatively, fluorescein-based indicators have also been described that avoid accumulation in the mitochondria, yet have a shorter wavelength with less optimal emission spectra. Several calcium ion indicators are described in: Haugland, R. P. Handbook of Fluorescent Probes and Research Products, 9th Ed, Molecular Probes: Eugene, Oreg., 2002, Chapter 20; Martin et al., Cell Calcium 2004, 36, 509-14; and Beierlein et al., J. Neurophysiol. 2004, 92, 591-599.
Free cytosolic calcium ions play a key role in many aspects of cellular signalling and regulation, and fluorogenic calcium indicators like Fluo-4 (Invitrogen Corp.) provide quantitative and spatial information on calcium gradients with microscopic and plate reader measurements. Currently, green channel indicators like Fluo-4 enjoy a centerpiece role in a variety of investigative methodologies and HTS assays at Gi, Go, and Gs coupled G Protein Coupled Receptors (GPCRs).
Shifting the excitation of the fluorescent calcium indicator toward longer wavelengths would be beneficial for imaging applications by making it possible to multiplex the dye with existing green fluorophores and fluorescent protein constructs. It could also improve readout in HTS assays by shifting to wavelengths where compound library autofluorescence is less of a problem.
A need exists for compounds which have the advantage of a longer wavelength and avoid localization in the mitochondria. Furthermore, a need exists for fluorogenic probes that are taken up by cells and provide sensitive detection of cytosolic metal ions, such as calcium, when in the cell. Here we report the development and evaluation of a novel fluorescent calcium indicators having better loading and response characteristics than existing longer-wavelength calcium indicators.